1. Field:
The invention is in the field of separating charged particles or ions, particularly macro molecules such as proteins, through chromatography.
2. State of the Art:
Various types of chromatography columns are in use today for a variety of purposes. One use of such columns is in the separation of proteins. A commonly used method for protein separation involves ion-exchange chromatography on silica based, cellulose based, or agarose based columns. With such a column, a solution containing the protein or proteins to be separated is passed through the column, and the electrically charged proteins are captured by the oppositely charged ion-exchange adsorbent material of the column.
Once the proteins are adhered to the column, the proteins are removed from the column, preferably selectively, by either displacement or elution methods. The elution methods are most commonly used and involve either changing the pH of the buffer solution passing through the column or increasing the salt concentration in the buffer solution or both. When the pH of the solution is changed, the electrical charges on the proteins or on the ion-exchange adsorbent material in the column are changed and the proteins are released by the column. With an increase in background salt in the column, the salts break the bonds between the proteins and the ion-exchange adsorbent material in the column to similarly release the proteins. As the pH level is gradually changed or as the salt level is gradually increased, the proteins having the smaller number of electrical charges, i.e., of bonds to the column, will generally be released first and those with the larger number of charges will be released later. This is because it is more likely that, as individual bonds are broken, all of the bonds of a protein having fewer bonds will be broken first to release that protein. Band spreading is a problem in elution chromatography and the large volume of effluent necessary to accommodate this spreading is particularly burdensome on large columns. Usually, the volume of effluent is markedly reduced in large scale separations at the cost of lower resolution. Therefore, where good selectivity is needed, small columns with small quantities of proteins must be used.
With ion-exchange displacement methods of protein separation, the salt concentration and buffer pH in the column are kept constant while displacer molecules are passed through the column. The displacer molecules are chosen to have a number of like electrical charges exceeding the number offered by the protein to be released. The displacers are also chosen to have a higher affinity for the column material than the targeted proteins. In this way, when a displacer is introduced into the column, all proteins having the same or lesser charges are released. Then a displacer with a larger number of charges is introduced into the column to release proteins having charges in the range between the proteins initially released and those having a number of charges equal to those of the displacer. This process can be repeated with various displacers of increasing number of charges, as desired, to selectively release proteins in the column. Displacement provides good selectivity of the proteins released. However, while ion-exchange displacement is used frequently with anion-exchange columns because of the availability of anionic displacers, it is difficult to use ordinary displacement methods when the proteins have predominantly positive charges and are therefore adsorbed on cation-exchange columns for which anionic displacers have no affinity. Cationic displacers are not generally available and where available are very limited in variety, so ordinary displacement is not now practical with cation-exchange columns.